Journal: PLoS Pathogens

Article Title: Exploitation of the Complement System by Oncogenic Kaposi's Sarcoma-Associated Herpesvirus for Cell Survival and Persistent Infection

doi: 10.1371/journal.ppat.1004412

Figure Lengend Snippet: (A) Relative expression levels of CD46, CD55 and CD59 mRNAs in uninfected and KSHV-infected endothelial cells. The level of mRNA expression was measured by RT-qPCR. GAPDH gene was used as a calibration control. The expression levels of uninfected cells were set as “1”. Results shown as means ± SD are representative from three independent experiments. * P<0.05, ** P<0.01 and *** P<0.001 by Student's t -test. (B) Total protein levels of CD46, CD55 and CD59 were examined by Western-blotting using antibodies to CD46, CD55 and CD59, respectively. (C) Surface protein levels of CD55 and CD59 on uninfected and KSHV-infected endothelial cells were analyzed by flow cytometry. (D–E) Representative illustration of detection of CD55 (D) and CD59 (E) in KS tumors. (a) Paraffin-embedded KS tumor sections with typical spindle cells were stained with antibodies to C55 and CD59. Representative areas are shown in the top right panel. Typical KS tumor spindle cells are indicated by red arrows while immune cells are indicated by black arrows. (b) Uninvolved adjacent tissues with endothelial cells and mononuclear cells were stained for CD55 and CD59 as positive controls. Representative mononuclear cells were indicated with black arrows. (c) Negative control staining with the isotype-matched control antibodies in the same KS tumors as (a). The scale bar is 10 µm.

Article Snippet: A mouse monoclonal antibody to CD59 (MEM-43/5) and rabbit polyclonal antibodies to C5b-9 (ab55811) and C3d (ab15981) were from Abcam (Cambridge, MA).

Techniques: Expressing, Infection, Quantitative RT-PCR, Control, Western Blot, Flow Cytometry, Staining, Negative Control

Journal: PLoS Pathogens

Article Title: Exploitation of the Complement System by Oncogenic Kaposi's Sarcoma-Associated Herpesvirus for Cell Survival and Persistent Infection

doi: 10.1371/journal.ppat.1004412

Figure Lengend Snippet: (A–B) Western-blot examination of CD55 (A) and CD59 (B) protein expression in TIME-KSHV cells with stable overexpression of CD55 and CD59. TIME cells were used as controls for the relative expression levels of CD55 and CD59. (C) Flow cytometry analysis of CD55 (left panel) and CD59 (right panel) cell surface expression on TIME-KSHV cells with stable overexpression of CD55 and CD59. TIME cells were used as controls for the relative expression levels of CD55 and CD59. (D) Quantification of C5b-9 deposition on TIME-KSHV cells with overexpression of CD55, CD59 or vector control. Box and whisker plots showing the mean (middle line) and 25–75 th percentiles (lower and top box lines) of the average C5b-9 deposited areas on TIME cells (n = 10), TIME-KSHV cells (n = 10), and TIME-KSHV cells with overexpression of CD55 (n = 10), CD59 (n = 10) or vector control (n = 10) following exposure to 10% normal human serum for 1 h. Top and lower lines indicate the maximal and minimal values. Ten images from each cell type shown in were analyzed for C5b-9-positive areas using the ImageJ software. The average C5b-9 staining area per cell was calculated by dividing the total pixels of C5b-9 staining in a microscopic field by the cell number. (E) Flow cytometry analysis of C3b deposition on TIME-KSHV cells with overexpression of CD55 (left panel) or CD59 (right panel). TIME cells and TIME-KSHV cells transduced with the vector were used as controls. (F) Overexpression of CD55 or CD59 abolished complement activation of STAT3 tyrosine phosphorylation in TIME-KSHV cells. STAT3 tyrosine phosphorylation was examined in cells cultured in 10% normal human serum for 24 h. (G) Overexpression of CD55 or CD59 abolished the enhanced cell survival in TIME-KSHV cells cultured in medium depleted of growth factors. Cells were cultured for 48 h in heat-inactivated or normal human serum in endothelial cell medium depleted of growth factors in the presence of the indicated serum, and live cells were determined. Results are means ± SD from three independent experiments with three repeats. * P<0.05 and ** P<0.01 by Student's t -test.

Article Snippet: A mouse monoclonal antibody to CD59 (MEM-43/5) and rabbit polyclonal antibodies to C5b-9 (ab55811) and C3d (ab15981) were from Abcam (Cambridge, MA).

Techniques: Western Blot, Expressing, Over Expression, Flow Cytometry, Plasmid Preparation, Control, Whisker Assay, Software, Staining, Transduction, Activation Assay, Phospho-proteomics, Cell Culture

Journal: Journal of Virology

Article Title: Analysis of Complement-Mediated Lysis of Simian Immunodeficiency Virus (SIV) and SIV-Infected Cells Reveals Sex Differences in Vaccine-Induced Immune Responses in Rhesus Macaques

doi: 10.1128/JVI.00721-18

Figure Lengend Snippet: Cellular CD59 expression and inhibition of SIV lysis. (A to C) Naive, uninfected rPBMCs were evaluated for CD59 expression via flow cytometry: PE-isotype control staining (A), CD59-PE staining (B), and CD59-PE staining (C) of PIPLC-treated cells. The percentages of CD4+ CD59+ cells from one representative animal are shown. (D) rPBMCs were collected from 25 macaques prior to vaccination (Pre; dotted bars) and following 8 weeks of SIV infection (PI; solid bars), and CD4+ T cells were evaluated for CD59 expression via flow cytometry. The average percentages of CD4+ CD59+ cells for males (blue) and females (red) at both time points are shown. Error bars represent SEM. (E) SIV was either treated with PIPLC or left untreated, and ADCML was assessed using sera collected from 15 gp120-immunized macaques at week 57. The symbols represent values from individual macaques, bars represent the average, and error bars represent SEM. Asterisks indicate a significant difference (**, P <0.01). (F to H) Human H9 cells were evaluated for CD59 expression via flow cytometry: PE-isotype control staining of uninfected H9 cells (F), CD59-PE staining of uninfected H9 cells (G), and CD59-PE staining of SIV-infected H9 cells (H). The percentages of live, CD59+ cells from one representative experiment out of 3 individual experiments are shown.

Article Snippet: CD59 staining and flow cytometry. rPBMCs (1.5 × 10 6 ) isolated from 25 macaques prior to vaccination and following 8 weeks of SIV mac251 infection were stained with a LIVE/DEAD Fixable Aqua Dead cell stain kit (catalog number {"type":"entrez-nucleotide","attrs":{"text":"L34966","term_id":"522209","term_text":"L34966"}} L34966 ) from Thermo Fisher and with the following antibodies: CD3-V450 (catalog number 560351, clone SP34-2) from BD Biosciences (San Jose, CA), CD4-Qdot-605 (catalog number CD4-Qdot-605, clone 19-Thy-5D7) from the Nonhuman Primate Reagent Resource, and CD59-phycoerythrin (PE) (catalog number GTX75576, clone MEM-43/5) from GeneTex (Irvine, CA) or the mouse IgG2b-PE isotype control (catalog number 555058, clone 27-35) from BD Biosciences.

Techniques: Expressing, Inhibition, Lysis, Flow Cytometry, Control, Staining, Infection

Journal: Journal of Virology

Article Title: Analysis of Complement-Mediated Lysis of Simian Immunodeficiency Virus (SIV) and SIV-Infected Cells Reveals Sex Differences in Vaccine-Induced Immune Responses in Rhesus Macaques

doi: 10.1128/JVI.00721-18

Figure Lengend Snippet: Env-specific IgG/IgM exhibit different ADCML capabilities in males and females. (A and B) Env-specific IgM binding titers were assessed in sera collected from all vaccinated macaques. gp120-specific (A) and gp140-specific (B) IgM binding titers are shown; error bars indicate SEM, and asterisks indicate significant differences between males and females (*, P < 0.05; **, P < 0.01). (C and D) Statistical correlations between ADCML of SIV and gp120-specific IgG binding titers (C) and IgM binding titers (D) are shown for all vaccinated females. (E to G) Statistical correlations between ADCML of SIV-infected H9 cells and gp140-specific IgG binding titers in all vaccinated animals (E) and gp120-specific (F) and gp140-specific (G) IgM binding titers in all vaccinated males are shown. The animal cohorts are displayed within each graph heading, and the symbols represent values from individual macaques. Significant correlations, calculated using Spearman's rank correlation coefficient, are indicated by r and P values and linear regression trend lines (added for visual aid).

Article Snippet: rPBMCs (1.5 × 10 6 ) isolated from 25 macaques prior to vaccination and following 8 weeks of SIV mac251 infection were stained with a LIVE/DEAD Fixable Aqua Dead cell stain kit (catalog number {"type":"entrez-nucleotide","attrs":{"text":"L34966","term_id":"522209","term_text":"L34966"}} L34966 ) from Thermo Fisher and with the following antibodies: CD3-V450 (catalog number 560351, clone SP34-2) from BD Biosciences (San Jose, CA), CD4-Qdot-605 (catalog number CD4-Qdot-605, clone 19-Thy-5D7) from the Nonhuman Primate Reagent Resource, and CD59-phycoerythrin (PE) (catalog number GTX75576, clone MEM-43/5) from GeneTex (Irvine, CA) or the mouse IgG2b-PE isotype control (catalog number 555058, clone 27-35) from BD Biosciences.

Techniques: Binding Assay, Infection

Journal: Journal of Virology

Article Title: Analysis of Complement-Mediated Lysis of Simian Immunodeficiency Virus (SIV) and SIV-Infected Cells Reveals Sex Differences in Vaccine-Induced Immune Responses in Rhesus Macaques

doi: 10.1128/JVI.00721-18

Figure Lengend Snippet: Systems serology approach to identify global sex differences in antibody profiles. (A to D) Box plots illustrate sex-specific levels, and scatter plots depict the sex-specific statistical correlations between SIV ACDML and IgG Gag SIVmac239 (A), glycoform G2S1B (B), glycoform G1′/G0FB (C), and glycoform G1′FB/G2 (D). For panels B to D, the G in the glycoform name indicates galactosylation, S indicates sialylation, F indicates fucose, and B indicates a bisecting glycoform. The numbers that follow G and S indicate the number of galactose and sialic acid molecules. The symbols represent values from individual macaques, and correlation r and delta r values are displayed. (E) Sex-specific correlation heatmaps illustrating all pairwise Spearman correlations between the measured analytes (Fc effector functions, glycosylation profiles, and FcγR binding affinities).

Article Snippet: rPBMCs (1.5 × 10 6 ) isolated from 25 macaques prior to vaccination and following 8 weeks of SIV mac251 infection were stained with a LIVE/DEAD Fixable Aqua Dead cell stain kit (catalog number {"type":"entrez-nucleotide","attrs":{"text":"L34966","term_id":"522209","term_text":"L34966"}} L34966 ) from Thermo Fisher and with the following antibodies: CD3-V450 (catalog number 560351, clone SP34-2) from BD Biosciences (San Jose, CA), CD4-Qdot-605 (catalog number CD4-Qdot-605, clone 19-Thy-5D7) from the Nonhuman Primate Reagent Resource, and CD59-phycoerythrin (PE) (catalog number GTX75576, clone MEM-43/5) from GeneTex (Irvine, CA) or the mouse IgG2b-PE isotype control (catalog number 555058, clone 27-35) from BD Biosciences.

Techniques: Binding Assay